Chemical gradients play an important role in mediating biological activity in vivo. Insight into the interplay between a chemical gradient treatment and the corresponding cellular response may help to determine the cues that trigger changes in gene expression that are responsible for regulating specific cellular activities. Understanding the importance of these chemical cues could help researchers develop controlled microenvironments wherein the desired cellular response is produced by combining the effects of exogenous controlled gradient treatments with ongoing endogeneous cell-cell signaling.
Prior to the development of laminar flow based gradient generators, it was difficult to accurately develop and predict the chemical microenvironment to which cells are exposed. Laminar flow based gradient generators create chemical gradients by taking advantage of diffusional mixing across the interface of adjacently flowing streams. With these gradient generators, it is possible to treat a cell population with a controlled chemical gradient and to observe the biochemical and morphological responses of the cell in vitro.
These prior gradient generators include continuously flowing streams of fluid that provide precise control over the stability, gradient profile, concentration range and slope of a chemical gradient. The stimulus of interest can be changed “on the fly” to create a sequential chemical gradient treatment scheme. Flow based gradient generators have been used to successfully study neutrophil chemotaxis and neuronal differentiation in vitro. While these gradient generators are robust and provide excellent control over the chemical gradient characteristics, the continuously flowing streams that are necessary to maintain chemical gradients make these devices unsuitable for addressing certain biological questions wherein soluble factors are important in regulating cell behavior.
One way that cells respond to chemical cues in their environment is by secreting signaling factors that either affect the secreting cell itself (autocrine), or affect other types of cells (paracrine). In flow based gradient generators, autocrine/paracrine factors of a cell cannot accumulate because the flowing fluid streams immediately carry the secreted factors away. In situations where cell-cell communication (via soluble factors) plays a critical role in regulating biochemical activity, the removal or accumulation of secreted factors may lead to distinctly different cellular behavior. In view of the foregoing, it can be appreciated that to provide a microfluidic gradient generator that does not require flowing fluid streams to develop a stable chemical gradient.
Therefore, it is a primary object and feature of the present invention to provide a microfluidic platform and a method of generating a gradient therein.
It is a further object and feature of the present invention to a microfluidic platform and a method of generating a gradient therein that does not require flowing fluid streams to develop a gradient.
It is a still further object and feature of the present invention to a microfluidic platform and a method of generating a gradient therein that allows for the introduction of media into the gradient without generating convection.
It is a still further object and feature of the present invention to provide a microfluidic platform and a method of generating a gradient therein that is simple to utilize and inexpensive to manufacture.
In accordance with the present invention, a microfluidic device is provided for generating a gradient. The microfluidic device includes a body defining a source and a gradient channel. The gradient channel has an input port and an output. A first membrane separates the input port of the gradient channel and the source. A second membrane is disposed downstream of the first membrane. A sink communicates with the output of the gradient channel.
The sink may include a flow channel extending through the body and the second membrane may be disposed adjacent the output of the gradient channel. Alternatively, the sink may include a chamber having a predetermined volume. The gradient channel has a predetermined volume that is less than the predetermined volume of the sink. A media addition port communicating with the sink may also be provided in the body. The second membrane may be disposed across the media addition port. It is contemplated for the membranes to be formed from a polycarbonate material.
In accordance with a further aspect of the present invention, a microfluidic device is provided for generating a gradient. The microfluidic device includes a body and a first membrane. The body defines a source channel extending along a first axis and having an output; a gradient channel at a predetermined angle to the source channel; and a sink communicating with the output of the gradient channel. The sink is defined by a flow channel extending through the body. The gradient channel includes an input communicating with the output of the source channel and an output. The first membrane extends through the source channel.
The gradient channel extends along a second axis that is generally perpendicular to the first axis. The sink may include a flow channel extending through the body or a chamber having a predetermined volume. The gradient channel has a predetermined volume that is less than the predetermined volume of the chamber of the sink. It is contemplated for the source channel to have an input operatively connected to a source of particles. The input of the source channel lies in a first plane and the gradient channel lies in a second plane axially spaced from the first plane.
In accordance with a still further aspect of the present invention, a method is provided for generating a gradient of particles within a microfluidic device. The microfluidic device defines a channel having an input and an output. The method includes the steps of filling the channel with a predetermined fluid and passing the particles through a porous first membrane into the channel. A second membrane is provided downstream of the first membrane to limit convection of the fluid in the channel.
A sink may be provided at the output of the channel. The sink includes a generally constant concentration of particles therein. The channel has a predetermined volume that is less than a predetermined volume of the sink. It is contemplated for a fluid stream to communicate with the sink. The method also contemplates passing the particles through a second porous membrane into the channel.